1. Field of the Invention
The present invention relates to a method of determining an antigen in a test specimen, and more particularly to an immunoassay which makes use of agglutination by a two-stage reaction using an insoluble carrier, has high specificity and is simple and low in cost.
2. Description of the Background Art
As immunoassays based on an antigen-antibody reaction, there have heretofore been known assays making use of an agglutination reaction and assays making use of an antibody labeled with an enzyme for detection. In these immunoassays, the amount of an immune complex formed by a specific antigen-antibody reaction is determined either visually or as an optical change. In particular, a method (hereinafter referred to as xe2x80x9cagglutination methodxe2x80x9d) of determining an antigen in a test specimen making use of an agglutination reaction or agglutination inhibition reaction based on the antigen-antibody reaction of insolubilized particles (hereinafter referred to as xe2x80x9cimmobilized antibodyxe2x80x9d) obtained by holding an antibody on an insoluble carrier with an antigen responsive to the antibody permits automating of determination, and so is applied to automatic analyzers and widely spreads.
Many of the agglutination methods heretofore in use employ latex particles as the insoluble carrier, and it is known to react an immobilized antibody using (1) a polyclonal antibody, (2) a kind of monoclonal antibody or (3) two kinds of monoclonal antibodies with an objective antigen in a test specimen to form an immune aggregate, and determine the degree of the agglutination either visually or optically. Besides, there is also known (4) a method in which an objective antigen in a test specimen is adsorbed on or bound to an insoluble carrier, and an antibody responsive to the antigen is then reacted to selectively agglutinate the insoluble carrier (Japanese Patent Application Laid-Open No. 35752/1995).
However, the above-described conventional methods involve the following drawbacks. Namely, the method of (1) is the most commonly used method but involves such problems that the polyclonal antibody cross-reacts to foreign antibodies derived from a trace amount of foreign components contained in an antigen used for antibody formation and to other components similar in structure to the objective antigen because the specificity of an assay system is affected by the specificity of the polyclonal antibody used. The method of (2) can be used only for special antigens in which there are a plurality of parts (hereinafter referred to as xe2x80x9crecognition sitesxe2x80x9d) which participate in the antigen-antibody reaction because only a kind of monoclonal antibody is used. According to the method of (3), an immune agglutinate is formed by increasing the number of recognition sites to a number corresponding to the number of antibodies by using two kinds of monoclonal antibodies. However, not that a combination of any two kinds of antibodies may be used so far as they are monoclonal antibodies responsive to the same antigen, but there is a problem that a combination of special two kinds of antibodies must be selected according to an object. Further, the method of (4) involves a problem that the insoluble carrier is non-specifically adsorbed on a reactor of an automatic analyzer, so that the reactor is contaminated.
There is also a method in which an immobilized antibody and a free antibody are used (Japanese Patent Publication No. 31227/1991). However, this method comprises, in a reaction system in which the immobilized antibody reacts to an object of determination to form an optically measurable immune agglutinate, causing both antibodies (the free antibody and immobilized antibody) to compete to the object of determination, thereby inhibiting the occurrence of immune agglutination to enlarge a measuring range. Therefore, this method is different in both principle and object from the present invention in which two antibodies different in form from each other are used in order to cause and increase immune agglutination.
In view of the above-described problems, the present invention has been made and has as its object the provision of an immunoassay which makes use of agglutination of an immobilized antibody to an object of determination and has high specificity.
In view of the foregoing circumstances, the present inventors have carried out an extensive investigation. As a result, it has been found that two kinds of antibodies, which respectively recognize different sites of an objective antigen of determination, are used and successively reacted in a state that one of them is immobilized, and the other is free, thereby permitting the achievement of an immunoassay which has high specificity and is simple and low in cost, thus leading to completion of the present invention.
According to the present invention, there is thus provided an immunoassay comprising reacting an immobilized antibody obtained by holding an antibody, which recognizes a part of an objective antigen of determination, on insoluble carrier particles with an antigen in a test specimen, then reacting a free antibody, which recognizes an antigen site different from that recognized by the immobilized antibody, with the antigen, and optically determining the degree of a change in agglutination occurred by the reaction.
According to the present invention, there is also provided an immunoassay comprising reacting a free antibody, which recognizes a part of an objective antigen of determination, with an antigen in a test specimen, then reacting an immobilized antibody obtained by holding an antibody, which recognizes an antigen site different from that recognized by the free antibody, on insoluble carrier particles with the antigen, and optically determining the degree of a change in agglutination occurred by the reaction.
The immunoassays according to the present invention have advantages that they have high specificity and are simple and low in cost, and with respect to the antibodies used, insofar as one of the immobilized antibody and the free antibody has high specificity for the objective antigen of determination, the other antibody does not need to have strict specificity and may have some cross-reactivity.
The above and other objects, features, and advantages of the present invention will be readily appreciated from the preferred embodiments of the present invention, which will be described subsequently in detail with reference to the accompanying drawings.